Neb monarch pcr dna cleanup kit

CUT&RUN were performed according to manufacturer’s instructions (EpiCypher CUTANA^™ pAG-MNase for ChIC/CUT&RUN, Cat# 15-1116). In brief, after washing with CUT&RUN wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× Roche Complete Protease Inhibitor), a million of cells were first bound to activated ConA beads (Bangs Laboratories, cat# BP531), followed by addition of anti-H4K20me3 antibody (Abcam, ab9053; 1:100 dilution) and cell permeabilization with the digitonin buffer (CUT&RUN wash buffer plus 0.01% digitonin). After washing in the digitonin buffer, samples were incubated with pAG-MNase, followed by additional washes with digitonin buffer. After the final wash, pAG-MNase activation was induced for DNA digestion by suspending cell samples in the pAG-MNase digestion buffer (digitonin buffer plus 2 mM CaCl₂) and incubation on nutator at 4 °C for 2 h. Solubilized chromatin was released using the stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/ml RNase A, 50 µg/ml glycogen) and collected using a PCR cleanup kit (New England BioLabs [NEB] Monarch PCR & DNA Cleanup Kit, cat# T1030). Ten nanogram of the purified CUT&RUN-enriched DNA was used for preparation of multiplexed Illumina libraries using the NEB Ultra II DNA Library Prep Kit according to manufacturer’s instructions (NEB cat#E7103).

For circularization, a 20-μl solution containing 2 μl of 10× T4 RNA Ligase Buffer (NEB), 0.4 μl of 10 mM ATP, 4 μl of 50% PEG8000 (NEB), 0.2 μl of T4 RNA Ligase 1 (NEB), 3.7 μl of Nuclease-Free water, and 10 μl of 20 amol/μl single-strand products was incubated at 37 °C for 16 h and 65 °C for 15 min to inactivate the enzyme. This reaction was performed in 8 tubes. In total, 240 μl of the circularized products were purified using the NEB Monarch PCR DNA clean up kit (NEB).